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Definition and Background of the Use-Dilution Test
The use-dilution test is a method of analyzing the efficacy of a liquid disinfectant in decreasing microbial counts on hard, nonporous, inanimate surfaces in the environment. A small stainless steel cylinder, usually measuring around 10 mm in length and 8 mm in diameter, is coated in a culture of a specific microorganism and allowed to dry. When the cylinder is dry, it is dipped in a series of disinfectant solutions for set amounts of time. The cylinder is then placed in a test tube of sterile medium and allowed to incubate. If microbes are present on the cylinder, the medium will appear turbid (cloudy), indicating that the disinfectant was ineffective at significantly reducing microbial numbers. If the medium appears clear following incubation, the disinfectant is likely effective at markedly reducing microbial numbers. Developed by the Association of Official Agricultural Chemists International (AOAC), a nonprofit group that sets many protocol standards in the field of chemistry, the use-dilution test must usually produce positive results in 59 out of 60 replications to establish a valid claim of the efficacy of the disinfectant, across multiple batches of disinfectant and multiple days of tests.
Applications of the Use-Dilution Test
The AOAC standards as described above have been adopted by numerous entities, from government agencies to private companies, for determining appropriate disinfectants to use in a variety of settings.
The Use-Dilution Test in Government Agencies
In 2020, the Environmental Protection Agency (EPA) and its Office of Pesticide Programs published a revised SOP (standard operating procedure) document describing the use-dilution method in testing disinfectants against three major microbes that commonly form biofilms (a group of bacteria that adhere together and to a surface): Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella enterica. The EPA uses the use-dilution test to assess the efficacy of antimicrobial pesticides on these biofilm-forming bacteria.
The Use-Dilution Test in Research
The use-dilution test can also be used to test disinfectants on viral pathogens, such as the Newcastle Disease Virus, which causes the reportable, contagious, and often fatal Newcastle Disease in poultry such as chickens. Finding an effective disinfectant for use in livestock settings such as a large layer or broiler farms is essential to inactivating any virus in the environment and preventing Newcastle Disease from gaining a foothold in the poultry industry. In 1964, researchers from the University of California modified the original use-dilution test, dating back to 1960, for use on viral microorganisms. They were able to produce useful results and determine that use-dilution tests are valuable for testing disinfectants on viruses and bacteria.
The Use-Dilution Test in Commercial Settings
There are multiple commercial product testing laboratories private companies can use to evaluate disinfectants for use in their specific settings or to market to other companies. For example, one such purveyor of testing services, Illinois-based Situ Biosciences, offers use-dilution tests accord to AOAC protocol for three bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella enterica) and one fungus (Trichophyton mentagrophytes). Situ Biosciences further allows customers to specify whether they are submitting use-dilution test results for research and development purposes or to comply with EPA regulations described above and to specify whether they would like the testing conditions to conform to GLP (Good Laboratory Practices) standards, which are more rigorous, or non-GLP standards.
As we have seen in the lesson, the use-dilution test, which employs small metal cylinders that have been treated with microbes, exposed to disinfectants at various concentrations for a prescribed amount of time, then incubated in sterile test tubes to measure microbial growth following culture, is a widely-applicable method for assessing the efficacy of liquid disinfectants on hard, nonporous surfaces. We examined three significant fields in which use-dilution tests are used for such a purpose. We first looked at government agencies, specifically the EPA, and how it uses the AOAC protocol to establish standards for testing antimicrobial pesticides. The use-dilution test was also viewed in a research setting, such as the University of California scientists who modified it for viral pathogens, namely the Newcastle Disease Virus. Finally, we learned that the use-dilution test is offered commercially by several private laboratories as a customizable testing service depending on client specifications and the test's purpose. Dating back to 1960, the use-dilution test has a history in disinfectant development, and it continues to be an important tool for producing improved disinfectants today.
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